Subject of this invention is a method for the detection of actual mutative changes in the genome DNA, whereby the possible mutation site has to be known beforehand. These mutative sequence changes may either be a base exchange (point mutation) or the introduction of nucleotides (insertion) or removal of nucleotides (deletion). Point mutations have become particularly well known under the abbreviation SNP (single nucleotide polymorphisms). For humans, is it supposed that there are at least 3 million frequently occurring SNPs which characterize most of the individual differences between humans. They control the individual phenotypes.
For the genome of a species, it is customary to define a “wild type” which is regarded as free of mutation, and a “mutant” which contains a mutation. Considering the frequency of mutations, such as SNPs, and the equal value of mutants and wild types, the definition of the wild type is arbitrary or at least purely accidental.
Nearly all DNA mutations, including all those defined above, produce differences in the mass of the DNA segment containing the mutation in comparison to the mass of a corresponding segment of the wild type. The precise mass determination of a DNA segment can therefore be used for the determination of a mutation.
Mass spectrometry is a very powerful and precise tool for determining the mass of a bio-molecule. By using a mass spectrometric method, such as time-of-flight mass spectrometry (TOF-MS) with ionization by matrix-assisted laser desorption and ionization (MALDI), it is possible to analyze the ions for their masses. However, ionization can also be achieved using electrospray ionization (ESI), in the latter case with mass spectrometers which are generally of a different type.
With polymerase chain reactions (PCR), using a pair of “selection primers”, i.e. single strand oligonucleotides about 20 bases long, it is possible to produce double-strand DNA products with a length of at least 40 base pairs in a known way. The mutation site can be incorporated by adequately choosing the sequences of the two selection primers.
The obvious method to simply measure the mass of the PCR-amplified DNA products as such by mass spectrometry, was found to be almost unworkable. The precise measurement of DNA products with more than 40 base pairs proved itself to be almost impossible. The reasons for this are given below. A method therefore had to be found to provide much shorter DNA fragments. To this end, the method of restricted, mutation-dependent primer extension by the use of terminating derivatives of the nucleotide triphosphates has been developed in order to generate extended primers of approximately 15 to 25 nucleotides in length, better suited to identify the nature of the mutation.
This method consists of the following steps: Firstly, a sufficient number of copies of the DNA segment containing the mutation site is produced by PCR using a pair of primary primers. These DNA segments then serve as templates for the enzymatic, mutation-dependent extension of a secondary primer. If in the following the word “primer” is used, always this secondary or extension primer is meant. The extension primer may be identical with one of the two selection primers; however it is regularly much better to use a primer which is not identical.
This primer is a short DNA chain of approximately 15 to 25 nucleotides and functions as a recognition sequence for the site of a possible mutation. The primer is synthesized with a base sequence so that it can be annealed to the template strand as an exact compliment to the base in the vicinity of a known point mutation site. (This form of attachment is known as “hybridization” or “annealing”).
In the simplest case, the primer is designed so that it is attached directly neighboring the possible mutation site. The enzymatically controlled extension of this primer by a polymerase is carried out using the dideoxy versions (ddNTP) of the four deoxynucleoside triphosphates (dNTP or, to be precise, dATP, dGTP, dCTP and dTTP). These dideoxynucleoside triphosphates serve as terminators; when built in, they terminate the extension. Which one of the four ddNTPs is inserted depends on the master template: the mutation site is mirrored in a complementary fashion. The four possible terminators differ (as do the associated deoxynucleoside triphosphates) by at least 9 and by a maximum of 40 atomic mass units. In principle, therefore, conclusions about the actual mutation can be made from the mass determination of the primer which has been extended in this way. This method, which always leads to products with the same number of nucleotides, will be referred to in the following as “equal-number-nucleotide primer extension”.
Unfortunately, precise determination of the mass of oligonucleotides is difficult. Because of the polyanion character of the DNA, various numbers of ubiquitous sodium (23 atomic mass units) or potassium ions (39 atomic mass units) are particularly likely to attach to the oligonucleotides (instead of the protons), and so-called “adducts” are formed. This uncertainty in the degree to which the adducts are formed makes any precise mass determination exceptionally difficult—at the very least, it means that cleaning has to be extremely thorough and all process parameters have to be carefully monitored for constancy. The equal-number-nucleotide primer extension method is therefore very difficult to use as it is susceptible to error.
Another method of mutation-dependent primer extension has therefore been developed in which the mass difference between the two homozygote forms of the DNA product, i.e. the wild type and the mutant, amounts to at least one nucleotide, i.e. at least approximately 300 atomic mass units. This method will be referred to in the following as “different-number-nucleotide primer extension”.
In this case, the primer does not have to hybridize directly next to the possible mutation site. Between the site of the possible mutation and the 3′ position of the primer (the position where the primer is extended), the sequence of the template strand may consist of three of the four nucleotides in maximum. The fourth nucleotide appears for the first time at the mutation site. By using, firstly, a polymerase and, secondly, a particular set of unmodified deoxynucleoside triphosphates (dNTP) complementary to the maximum three nucleotides which bridge the 3′-end of the extension primer and the mutation site, and, thirdly, at least one dideoxynucleoside triphosphate (ddNTP) complementary to the fourth type of nucleotide, the primer is extended as a complementary copy at the template. The chain extension is terminated by the dideoxynucleoside triphosphate. Depending on whether a point mutation is present or absent, the polymerase reaction is terminated at the mutation site or is not terminated until the next nucleotide corresponding to a terminator on the other side of the potential mutation site. The extension products of wild type and mutant differ after this different-number-nucleotide primer extension, i.e. they differ in length by at least one nucleotide or by at least approximately 300 atomic mass units. Thus, high mass precision is no longer necessary for determining the mutation type; the difference between the masses of the mutation and the wild type are beyond the range of metal-ion adducts. The mass spectrometric analysis is therefore made considerably easier.
This method of different-number-nucleotide primer extension requires somewhat more elaborate preparation since each of the four types of nucleobases at the mutation site requires its own mixture of dNTPs and ddNTPs. Another disadvantage is the lower degree of multiplexing possible: the number of mutations which can be detected in a single sample at the same time is lower than that for the equal-number-nucleotide primer extension method initially described above. Multiplexing capability is also restricted by the fact that a compatible set of dNTPs and terminators must be found for each reaction to be combined. However, the method has the advantage of being more insensitive to traces of impurities and to changes in the measurement conditions. A further advantage is that there is greater freedom in selecting a site of attachment and this means that the same process conditions for extension can in most cases be maintained because the primer can be designed with greater freedom of choices.
The MALDI preparation and measurement procedure consists of first embedding on a sample support the analyte molecules in a solid UV-absorbent matrix, usually an organic acid. The sample support is then introduced into the ion source of the mass spectrometer. The matrix is then evaporated by a short laser pulse of about 3 nanoseconds, producing a so-called plume consisting of a weakly ionized plasma. This process transports the analyte molecules into the gas phase but, unfortunately, a part of the fragile analyte molecules will be fragmented. The analyte molecules are ionized as a result of collisions with matrix ions of the plume. The voltage which is applied to the ion source apertures accelerates the ions into the flight tube which has no electrical field. Due to their differing masses, the ions are accelerated to different velocities. The smaller ions reach the detector earlier than the larger ions. The flight times are measured and converted into ion masses.
MALDI is ideally suited for the analysis of peptides and proteins. The analysis of nucleic acid chains is somewhat more difficult. Ionization in the case of short-chain nucleic acids in the MALDI process is approximately 100 times less intense than it is for peptides and decreases superproportionally with increasing mass. The reason for this is that only a single proton has to be captured to ionize peptides and proteins. For nucleic acids with multiple negative charges on the poly-anionic sugar-phosphate backbone (one negative charge for each nucleotide), the process to generate singly charged ions has to use such a lot of protons that it is considerably less efficient. The DNA segments (extended primers) which have to be detected must therefore be as short as possible so that they can be detected well.
In a similar way, an ionization method can also be used which uses a solution of the samples as the starting point. This is known as electrospray ionization (ESI). The method is also ideally suited to the detection of peptides and proteins but has similar problems with oligonucleotides. Here also, the oligonucleotides which are to be detected have to be as short as possible.
In MALDI, the choice of the matrix substance plays an important role. There are quite a number of very efficient matrices for the desorption of peptides. Up to now, however, there are only a few efficient matrices for DNA, and they do not solve the sensitivity and adduct problems for DNA.
However, this sensitivity difference can be reduced by chemically modifying the DNA so that it more resembles a peptide. As explained in WO 96/27681 (Gut and Beck), phosphorothioate nucleic acids, for example, where the phosphate groups are replaced by thiophosphate groups, can be converted into a neutrally charged DNA by simple alkylation. This neutrally charged modified DNA can be ionized like a peptide. Adduct formation and fragmentation hardly occur. This modification has made it possible to use matrices which are similar to those used for the desorption of peptides.
In addition to the neutralization, it is also possible to bond a group with a single positive or negative charge (charge tag) covalently to this modified DNA. Attaching a charge means that non-protonating matrices can be used. The use of non-protonating matrix substances leads to an increase in sensitivity for the modified DNA and suppresses the ionization of impurities not carrying a charge. Another advantage of “charge tagging” is the increased stability of the analysis as far as the impurities are concerned which make the detection of unmodified DNA analyte molecules much more difficult.
But even for these modified oligonucleotides, it has been found to be beneficial to keep the segments which are to be analyzed as short as possible.
Using phosphorothioate nucleic acids, a method has been developed for shortening the oligonucleotides whereby a part of the extended primer is digested using an enzyme. This can be achieved by providing only a short chain of nucleotides at the 3′ position of the extended primer with thioate groups and not modifying the majority of the nucleotides. The regular nucleotides can then be digested using an exonuclease (e.g. phosphodiesterase) whereas the phosphorothioates are resistant to digestion. Exonuclease digestion takes a considerable amount of time and is not always quantitative.
Another method to shorten the products which have to be analyzed mass spectrometrically was proposed by Monforte et al. (J. A. Monforte, C. H. Becker, T. A. Shaler, D. J. Pollart, WO 96/37630). The authors introduce linkers into the primers which can be cleaved chemically or enzymatically. The introduction of chemicals, however, always has the disadvantage of introducing traces of impurities which again may form adducts. In addition, chemical cleavage needs adjustments of other parameters of the solution as for instance pH values, needing more chemicals to be added with the danger to introduce, e.g., alkali ions. Enzymatic cleaving, e.g. by restriction endonucleases, means a very restricted design of the primers which have to offer a recognition pattern for the nucleases and also needs adjusted buffer conditions for cleavage, making washing after cleavage a necessary step.
Sample preparation methods for mass spectrometric mutation analyses which are simpler and more reliable are therefore still being sought.